Green fluorescent protein is widely used in protein localization and protein dynamics analysis. GFP Magnetic Beads are conjugated with GFP-tag antibodies and are specifically used for purifying and detecting GFP-tagged proteins in cell lysates or bacterial lysates. They are suitable for IP experiments. When the cell or bacterial lysate is mixed with this reagent, the GFP-tagged recombinant proteins in the sample specifically bind to GFP Magnetic Beads, while other contaminants do not. This reagent has a stable structure and high efficiency in binding to GFP-tagged proteins. The IP products can be applied in biochemical analysis methods such as mass spectrometry analysis and enzyme activity determination. This product is a specially prepared GFP beads product. When using this product for IP experiments, in the subsequent IP-WB steps, the appearance of bands from the light and heavy chains of the antibody can be completely avoided.

IP

All Species Expected

Transfected Only

Anti-GFP Tag mAb

IP 1:50

Mouse IgG

The beads are recommended to be stored at 4℃. The product is stable for 12 months at 4℃. Freezing the magnetic beads will irreversibly damage the bead structure.

Lysis buffer (IP、CoIP)：10mM Tris-HCl pH 7.5;150mM NaCl;0.5mM EDTA;0.5% NP-40

RIPA buffer：10mM Tris-HCl pH 7.5;150mM NaCl;0.5mM EDTA;0.1% SDS;1% Triton X-100;1% Deoxycholate

Dilution/Wash buffer：10mM Tris-HCl pH 7.5;150mM NaCl;0.5mM EDTA

2×SDS loading buffer：120mM Tris-HCl pH 6.8; 20% glycerol; 4% SDS, 0.04% bromophenol blue; 10%β-mercaptoethanol

AB_2936245

Experimental Procedure for Label Antibody-Conjugated Magnetic

1. Cell Harvesting

For a single immunoprecipitation reaction, it is recommended to use 10⁶-10⁷ mammalian cells expressing the GFP fusion protein.

Remove the culture medium, add 1mL of pre-chilled PBS to the cells, and scrape the cells off using a cell scraper.

Transfer the cells to a pre-chilled centrifuge tube.

Centrifuge at 4°C, 500×g for 3 minutes, then discard the supernatant. Wash the cell pellet twice with pre-chilled PBS and gently resuspend the cells.

2. Cell Lysis

Resuspend the cells in 200μL of pre-chilled Lysis Buffer.
Note: Protease inhibitors (cocktail type recommended, Cat. No. A10004: 200×Protease Inhibitor Cocktail) must be added to the Lysis Buffer.

Place the centrifuge tube on ice for 30 minutes, and resuspend the cells every 10 minutes to ensure complete lysis.

Centrifuge at 4°C, 16,000×g for 10 minutes.

Transfer the supernatant to a new pre-chilled centrifuge tube, add 300μL of Dilution Buffer, and discard the pellet.
Note: The cell lysate obtained in this step can be stored long-term at -80°C. Protease inhibitors must also be added to the Dilution Buffer.

3. Magnetic Bead Equilibration

Vortex the magnetic beads to mix thoroughly.

Pipette 25μL of beads into a centrifuge tube containing 500μL of pre-chilled Dilution Buffer.

Centrifuge at 4°C, 1,200×g for 2 minutes, discard the supernatant, and repeat this operation twice to complete bead equilibration.

4. Protein Binding

Add the cell protein extract obtained in Step 2 to the equilibrated magnetic beads. Recommendation: Reserve 50μL of the supernatant as an input control for subsequent Western Blot analysis.

Incubate the centrifuge tube by inverting it up and down at 4°C for 1 hour to ensure sufficient binding between the beads and the target protein.

Centrifuge at 4°C, 1,200×g for 2 minutes, then discard the supernatant.

5. Magnetic Bead Washing

Resuspend the magnetic beads in 500μL of pre-chilled Lysis Buffer. Centrifuge at 4°C, 1,200×g for 2 minutes, discard the supernatant, and repeat the washing 2-3 times.

Optional Step: During the second wash, the salt concentration in the Lysis Buffer can be increased to 500mM to enhance the washing effect.

6. Bound Protein Elution

a. Routine Elution (for SDS-PAGE)

Resuspend the beads in 100μL of SDS Loading Buffer.

Heat the beads at 95°C for 10 minutes to dissociate the immune complexes.

Centrifuge at 4°C, 1,200×g for 2 minutes, and collect the supernatant for SDS-PAGE analysis.

b. Elution for Mass Spectrometry Identification (Alternative to Routine Elution)

Add 50μL of 0.2M glycine-HCl solution (pH 2.5) to resuspend the beads, and incubate with continuous mixing for 30 seconds.

Centrifuge at 4°C, 1,200×g for 2 minutes. Transfer the supernatant to a new centrifuge tube, and add 5μL of 1M Tris base (pH 10.4) to neutralize the supernatant.
This operation can be repeated to improve elution efficiency. Use the eluate for subsequent mass spectrometry identification.

7. Subsequent Experiments

The eluted supernatant can be used for experiments such as SDS-PAGE, Western Blotting or mass spectrometry identification.